Hematopoietic stem cells (HSCs) are rare cells within blood forming tissues that provide the life-long regeneration of all blood cell lineages. The absolute significance of proper HSC development can be seen in the incidence of numerous diseases that are caused by mutations in genes necessary for the correct hematopoietic gene regulation and blood cell development. One of these genes is the runt-related transcription factor 1 (Runxl), often a target of chromosomal translocation resulting in human leukemia. The complex expression pattern of Runxl is attributed to the presence of two distinct promoter regions, each driving the transcription of one Runxl isoform. The zebrafish will be used as a logical vertebrate model organism to study the Runxl isoforms and their biological functions in vivo. The main objective of this proposal is to investigate the roles of Runxl, taking a particular attention to its two promoters, in hematopoiesis beyond what has already been published. After the cloning of the distal and proximal Runxl isoforms, the differential expression patterns will be analyzed by several methods: semi-quantitative RT- PCR, whole mount in situ hybridization (WISH), and fluorescent-in situ hybridization (FISH). These experiments will provide a clearer picture of the significance of Runxl in the temporal and spatial regulation of HSCs. The functions of the Runxl isoforms will be further dissected by performing Runxl gain-of- function (by mRNA injection or heat-shock inducible transgenesis) and loss-of-function (by morpholkino injection) experiments. These experiments will be performed in conjuction with several already-established fluorescent transgenic zebrafish to investigate the effects of the Runxl isoforms on the lymphoid, myeloid, erythroid, and hematopoietic stem cells. To complement the morpholino analyses, a putative Runxl genetic mutant called thunderbird will also be used and analyzed. Finally, a triple trangenic line combining the often-used murine Cre/LoxP strategy and the zebrafish fluorescent trangenesis system will be generated. In this system, Runxl can be permanently overexpressed in a specific hematopoietic cell and its clonogenically-derived progeny throughout the life of the animal. The effects of constant Runxl expression can be studied in blood development and the occurrence of cancer and leukemias. Since Runxl is frequently involved in leukemias, the differential expression patterns of the two Runxl isoforms are clinically relevant. The proposed experiments will give us more clues as to the roles of Runxl in the development of hematopoietic stem cells. Furthermore, the use of the zebrafish as a vertebrate model system will provide a tool to perform high throughput drug and chemical screening to cure leukemia.